JP4759267B2 - Tumor marker proteins and uses thereof - Google Patents

Abstract

The invention relates to tumour marker proteins and their preparation from fluids from one or more cancer patients, wherein said fluids are those which collect in a body cavity or space which is naturally occurring or which is the result of cancer or medical intervention for cancer. Exemplary fluids are ascites, pleural effusion, seroma, hydrocoele and wound drainage fluid. The invention also relates to preparation of tumour marker proteins from excretions taken from patients with cancer. The said tumour marker proteins are useful in cancer detection methods which involve detecting or quantitatively measuring autoantibodies to circulating tumour markers or markers expressed on or in tumour cells and in various research applications. The invention is also directed to such uses.

Description

Field of Invention

The present invention relates to tumor marker proteins and their preparation from body fluids of one or more cancer patients. The body fluid, resulting in medical practice or cancer or cancer naturally occurring are those gathered in the body cavity. Exemplary body fluids are ascites, pleural effusion, seroma, varicella and wound drainage fluid. The present invention also relates to the preparation of tumor marker proteins from excreta collected from patients suffering from cancer.

  The tumor marker protein is useful for cancer detection methods including detecting or quantitatively measuring autoantibodies against circulating tumor markers or markers expressed on or in tumor cells, and various research applications. is there. The present invention is also aimed at such uses.

Background of the Invention

  Cancer development and progression in patients is generally found to be related to the presence of markers in the patient's body fluids, and these “tumor markers” reflect various aspects of cancer biology (See Fateh-Maghadam, A. & Steilber, P. (1993) Sensitive use of tumor markers., Verlag GMBH publication, ISBN 3-926725-07-9). Tumor markers are often found to be modified forms of wild-type proteins expressed by “normal” cells, which modifications include changes in primary amino acid sequence, changes in secondary, tertiary or quaternary structure, Or it may be a post-translational modification change, such as aberrant glycosylation. In addition, wild-type proteins that are up-regulated or over-expressed in tumor cells that may be the result of gene amplification or abnormal transcriptional regulation can also be tumor markers.

  Established assays for tumor markers present in body fluids tend to focus on the detection of tumor markers that reflect tumor mass and are therefore valuable in late stages of the disease process, for example in the diagnosis of metastatic disease . Among these markers, the most widely used are mainly carcinoembryonic antigen (CEA) and a glycoprotein called CA15.3, which were mainly useful as indicators of systemic disease burden and recurrence after treatment (Molina, R., Zanon, G., Filella, X. et al., Use of serial carbinoembryonic antigen and CA15.3 assays in detecting relaxes in breast cancer, 19). 41-48). These markers have limited use in the early stages of disease, such as early detection or screening of asymptomatic patients. In this way, during the search for tumor markers present in body fluids useful for assisting diagnosis early in the disease process, we attempted to identify markers that are independent of the tumor mass itself. .

  The difference between the wild-type protein expressed by “normal” cells and the corresponding tumor marker protein, in some cases, leads to the tumor marker protein being recognized as “non-self” by the individual's immune system, and thus An immune response can be elicited in the individual. This may be a humoral (ie B cell mediated) immune response that results in the production of autoantibodies that are immunologically specific for the tumor marker protein. An autoantibody is a naturally occurring antibody directed against an antigen that recognizes that the individual's immune system is foreign, even though the antigen is actually derived from that individual. It can be present in the circulation as a circulating free autoantibody or in the form of a circulating immune complex consisting of a combination of the autoantibody and its target tumor marker protein.

  As an alternative to direct measurement or detection of tumor marker proteins in body fluids, assays can be developed to measure an individual's immune response to the presence of tumor marker proteins with respect to autoantibody production. Such an assay essentially constitutes indirect detection of the presence of a tumor marker protein. Due to the nature of the immune response, autoantibodies are likely to be elicited by very small amounts of circulating tumor marker proteins, so that indirect methods that rely on detection of immune responses against tumor markers are in bodily fluids. It is more sensitive than the method of directly measuring tumor markers. Thus, assay methods based on the production of autoantibodies can be used in the early stages of the disease process, and in some cases in connection with screening of asymptomatic patients, eg, “risk of developing disease from a population of asymptomatic individuals. Can be particularly valuable in screenings to identify “in” individuals. Furthermore, they can be useful for early detection of recurrent disease.

  Tumor marker proteins that have been observed to induce serum autoantibodies are described in US Pat. No. 5,652,115, which can be defined by their ability to bind to the 70 kd heat shock protein (hsp70). Specific classes of mutant p53 proteins are included. p53 autoantibodies can be detected in many different benign and malignant patients (described in US Pat. No. 5,652,115), but in each case only a small subset of patients. not exist. For example, one study utilizing an ELISA assay to detect autoantibodies to p53 protein in breast cancer patient serum produced p53 autoantibodies in 26% of patients and 1.3% of control subjects. (Mudenda, B., Green, JA, Green, B. et al., The relations between serum p53 autoantibodies and charactaristics of human breast, 44, 49, 69, 94, 94). . A second tumor marker protein known to induce serum autoantibodies is epithelial mucin MUC1 (Hinoda, Y. et al. (1993), Immunol Lett., 35: 163-168; Kotera, Y. et al. Et al. (1994), Cancer Res. 54: 2856-2860).

  International Publication No. WO 99/58978 describes a method used for the detection / diagnosis of cancer based on the assessment of an individual's immune response to two or more different tumor markers. These methods generally involve contacting a sample of body fluid collected from an individual with a panel of two or more different tumor marker antigens, each derived from a separate tumor marker protein, and immunologically specific for the tumor marker protein. Detecting the formation of a complex of tumor marker antigens bound to various circulating autoantibodies. The presence of such circulating autoantibodies is used as an indicator of the presence of cancer.

  Cancer detection methods based on the detection of circulating autoantibodies are often immunoassays that utilize "immunoassay reagents" that are reactive with circulating autoantibodies. Typically, “reagents” for use in such assays include recombinant tumor marker proteins (expressed in bacterial, insect, yeast or mammalian cells) or chemically synthesized tumor marker antigens, which are substantially A complete tumor marker protein, or a fragment thereof such as a short peptide antigen. Other potential tumor-related protein sources used as a basis for immunoassay reagents to detect anti-tumor autoantibodies include cultured tumor cells (and spent media used for their growth), tumor tissue, and oncology Serum from an individual having Most of these sources have significant drawbacks as described below.

  In cultured tumor cells (and their spent media), the amount of protein expressed will vary depending on the growth phase at the time of recovery, and variations in quantity and quality may occur. Furthermore, the desired protein is generally present at a low concentration and therefore it takes time to purify a sufficient amount of protein. In addition, unlike cell stocks in tumors that are likely to be heterogeneous during neoplastic growth, cell stocks become clonal, which results in protein variability (particularly the degree of glycosylation).

  Recombinant proteins expressed in bacterial cells are not glycosylated and therefore they are significantly different from naturally glycosylated proteins. In addition, refolding of recombinantly expressed proteins may not be valid, thereby giving an incorrect conformation for autoantibody recognition.

  Tumor tissue is usually available only in small quantities, and it is difficult and time consuming to purify proteins from it.

  Serum samples are usually available only in small quantities, and it is therefore difficult to purify a sufficient amount of protein.

The inventors have now, ascites, pleural effusion, seroma, such Mizukobu or wound drainage fluid, tumor is present, or tumor associated with that or related to have body 腔由 come body fluids or excreta The significant advantages obtained by the use of tumor marker antigens purified from product-derived body fluids as “reagents” in autoantibody immunoassays were determined. Specifically, the present inventors have found that by using a reagent comprising a tumor marker antigens purified from the above defined body 腔由 come fluid, increase in sensitivity is caused (a reagent from a "normal" body fluid It was observed that more “clinically relevant” results were obtained than when used. There are also significant practical advantages obtained by using such bodily fluids as assay reagent sources.

Summary of the Invention

In a first aspect, the present invention is a method for detecting cancer-related anti-tumor autoantibodies, wherein a sample to be tested for the presence of such autoantibodies is contacted with an immunoassay reagent, and An immunoassay comprising detecting the presence of a complex formed by specific binding to any cancer-related antitumor autoantibodies present in a sample, wherein the immunoassay reagent comprises one or more cancers patients, the tumor had to or present there, or tumor associated with that or related to have body 腔由 come tumor marker protein prepared from bodily fluids, and / or one or excretion of more cancer patients The present invention relates to a method comprising a tumor marker protein prepared from a product, wherein the tumor marker protein exhibits selective reactivity with a cancer-related antitumor autoantibody.

In a second aspect, the invention provides for the presence or presence of a tumor in one or more cancer patients in the manufacture of an immunoassay reagent that exhibits selective reactivity with a cancer-related antitumor autoantibody. or tumor related to that or related prepared from the body 腔由 come fluid had tumor marker proteins, and / or to the use of tumor marker protein prepared from one or excreta of a plurality of cancer patients.

In a third aspect, the present invention provides a method for preparing a tumor marker protein comprising isolating the tumor marker protein from a body fluid, the body fluid comprising:
(I) the tumor had to or present there, or tumor are those related to that or related to have body lumen or al harvested and (ii) the body fluid is above 2 cancer patients Pool The present invention relates to a method for representing a body fluid sample.

In a fourth aspect, the invention provides a method for preparing a tumor marker protein comprising isolating said tumor marker protein from excreta,
(I) The excrement or any component thereof has been in contact with a tumor or tumor cells, and (ii) the excrement represents a pooled excrement sample of two or more cancer patients.

  In a fifth aspect, the present invention relates to a tumor marker protein preparation substantially free of immunoglobulins prepared using the method described above, and kits and reagents comprising said preparations.

Detailed Description of the Invention

  In a first aspect, the present invention relates to a method of detecting “cancer-related” anti-tumor autoantibodies.

  The term “cancer-related” anti-tumor autoantibody refers to an autoantibody directed to an epitope present on a tumor marker protein type that is characteristic of a cancer pathology and is preferentially expressed in the cancer pathology. Say.

The methods of the invention include immunoassays for detecting and / or quantitatively measuring autoantibodies that are immunologically specific for one or more tumor marker proteins, and are used in immunoassays. reagent "is, one or a plurality of cancer patients, the tumor had to or be present there, or tumor marker protein tumor was prepared from body fluids of the body 腔由 come you were or related are related, and / Alternatively, it comprises a tumor marker protein prepared from the excrement of one or more cancer patients. In general, the excreta should pass through the organ in which the cancer is present, where the excreta contacts the cancer, or the excrement contains one or more components that have contacted the cancer elsewhere in the body It becomes. A specific example is bile that can come into contact with cancer in the gallbladder but appears in the stool.

  Immunoassay reagents exhibit “selective reactivity” with cancer-related anti-tumor autoantibodies. As used herein, “selective reactivity” refers to any antibody or autoantibody generated against the same antigen that is present in a normal, i.e., tumor-free, condition. In comparison, it means having a higher affinity for autoantibodies against tumor-associated antigens.

The term "body cavity" includes any lumen or space exists in nature, including the collapsed lumen, or previous space, whether resulting lumen of the disease or medical practice, include any body lumen. The body fluid is from the cavity or space in which the tumor was or was present, or to which the tumor was or was associated. Preferably, a “body fluid derived from a body cavity” is a tumor-inducing body fluid, ie, a body fluid produced in response to or as a result of a disease process, eg, tumor cells. Examples of body fluids are ascites, pleural effusion, seroma, varicella and wound drainage fluid.

For the avoidance of doubt, in the "body 腔由 come of body fluid", body fluid from the systemic circulation, such as whole blood or serum it is not included.

  The term “feces” includes urine, feces, and semen, among others.

  The general features of immunoassays such as ELISA, radioimmunoassay are well known to those skilled in the art (see Immunoassay, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996). Immunoassays for detecting antibodies with a specific immunological specificity (eg, autoantibodies that are immunologically reactive with a given tumor marker protein) generally have specific immunology with the antibody under test. Requires the use of reagents that exhibit chemical reactivity. Depending on the form of the assay, this reagent can be immobilized on a solid support. A sample to be tested for the presence of antibody is contacted with a reagent, and if an antibody of the required immunological reactivity is present in the sample, these react immunologically with the reagent to form an autoantibody-reagent complex. Can be formed and then detected or measured quantitatively.

Suitable samples of tumor marker protein for use as the basis of the "immunoassay reagent", from one or more body 腔由 come cancer patients body fluids and / or one or more of body fluid from excretions of cancer patients Can be isolated using standard protein purification techniques, such as those commonly known in the art. For example, a tumor marker protein can be isolated by affinity chromatography using an appropriate antibody (or antibody fragment) immunologically specific for the tumor marker protein. The inventors have shown in the accompanying examples that several different tumor marker proteins can be purified using purification methods based on affinity chromatography. It will be apparent to those skilled in the art that similar purification methods used for any other tumor marker protein can be used using appropriate antibodies or antibody fragments.

  Body fluid starting material from body cavities and / or excreta is collected from one or more cancer patients. In this context, the term “cancer patient” includes individuals who have been previously diagnosed as having cancer. The body fluid / excretion may be collected from a single patient or two or more patient samples may be pooled together. Samples may be pooled from two or more patients suffering from the same or different stages of the same or different types of cancer. Samples can also be pooled from different types of bodily fluids or excretions from single or multiple patients. Advantageously, immunoassay reagents prepared from body fluids and / or excreta collected from cancer patients (or patients) suffering from a particular type of cancer are used to detect the same type of cancer in other individuals. Can assist with diagnosis.

  In one embodiment, the “cancer patient” from whom fluid / excretion is collected may be the same patient that is intended to be later tested with assay reagents. For example, a reagent stock prepared from a patient diagnosed with cancer may later be used to evaluate the immune status of the same patient, for example to monitor disease progression and / or the course of anticancer therapy in that patient. Can be used to assess sex.

  An “immunoassay reagent” or “tumor marker preparation” contains substantially the entire tumor marker protein, eg, the tumor marker protein that remains substantially isolated from the body fluid / excretion, or of the tumor marker protein. It may contain fragments. In order to be effective as an immunoassay reagent, all such “fragments” must retain immunological reactivity with the (auto) antibodies desired to be tested with the reagent. Appropriate fragments can be prepared, for example, by chemical or enzymatic cleavage of isolated tumor marker proteins.

  Depending on the detailed nature of the immunoassay used, the “reagent” or “tumor marker protein preparation” is linked to one or more additional molecules that provide certain desirable characteristics that are not naturally present in the tumor marker protein, Tumor marker proteins or fragments thereof may be included. For example, a tumor marker protein can be conjugated with a display label such as a fluorescent label, a color label, a luminescent label, a radiolabel or a heavy metal such as a gold colloid.

  Tumor marker proteins prepared by the methods described herein can also be immobilized for use on a solid support such as the surface of a plate of a bead or multiwell plate. Immobilization can be by absorption or covalent bonding.

  Tumor marker proteins (or assay reagents containing such proteins) are preferably substantially, thanks to processing of the protein preparation to specifically remove contaminating immunoglobulin, for example after isolation by affinity chromatography. Does not contain immunoglobulins.

  Use of an immunoassay reagent comprising a tumor marker protein (or a fragment thereof) isolated from body cavity fluid and / or excreta collected from one or more cancer patients can be used for clinical detection of cancer (diagnosis, disease recurrence or disease). Provides a significant advantage over the use of other reagents such as recombinantly expressed or chemically synthesized polypeptides.

  It can be expected that the detailed characteristics of a tumor marker protein isolated from a cancer patient may vary depending on the source material (eg, tissue or fluid) from which the tumor marker protein was isolated. For example, the characteristics of proteins isolated from urine may differ from those isolated from whole blood or serum, and these may also differ from proteins isolated from ascites or pleural effusions. In turn, this can affect the availability of tumor marker proteins as assay reagents.

  In fact, surprisingly, we generally have reagents prepared from tumor marker proteins isolated from body fluids or excretions from body cavities of cancer patients, particularly ascites, pleural effusion, seroma or wound drainage fluid, We observed higher specificity for cancer-related autoantibodies than reagents based on comparable proteins isolated from “normal” individuals. This increased specificity for cancer-related autoantibodies is more “clinically relevant” in the detection of immune responses to cancer by immunoassays based on the use of reagents prepared from body fluids or excretions from body cavities of cancer patients. This means that a “with” result will occur.

Prior to the present invention, it was unclear how reagents containing antigens prepared from body fluids or excretions from body cavities of cancer patients function as reagents for immunodetection of autoantibodies. Specifically, it was unclear whether such antigens showed higher specificity for cancer-related autoantibodies. It could not be predicted whether antigens from such sources would function as well or better than antigens prepared from blood or serum with respect to their ability to detect cancer-related autoantibodies. Although tumor marker protein has been known that may be present in body fluids come body 腔由, it is generally more likely that antigens of the body cavity is degraded. In turn, this means that they may not detect autoantibodies as well as serum-derived antigens. Furthermore, it was not possible to conclude reliably whether antigens derived from cavity-derived body fluids and feces were immunologically similar to serum-derived antigens. Thus, the observation that antigens prepared from body fluids and excretions from the cavity of cancer patients functioned as well as immunoassay reagents was surprising.

The present inventors have ascites, pleural effusion, seroma or wound drainage fluid improved specificity observed in the use of reagents prepared from body cavity-derived fluids of cancer patients, such as, such as the patient's body cavity Insist on the origin of body fluids. It is claimed to bodily fluid tumors in contact with the major organs may originate from body lumen by being present may pick up more tumor marker proteins "cancer-associated" type, in fact this is It is associated with cancer pathology and includes fewer corresponding “normal” proteins. Since it is generally the difference between a “tumor” marker protein and its “normal” counterpart that triggers the development of an immune response (ie, the production of autoantibodies), we have isolated tumor markers from cancer patients. Suppose that the reagent based on the use of is more specific for cancer autoantibodies than an equivalent “normal” protein. This is indeed the case with tumor marker antigens isolated from ascites, pleural effusion or seroma, as shown in the accompanying examples.

  There are further practical advantages associated with using ascites, pleural effusion, seroma, varicella or wound drainage fluid as a tumor marker protein source. These body fluids can easily be removed from the patient in a relatively large volume as part of a treatment strategy. This material that would otherwise be discarded is a valuable source of useful assay reagents.

  Given that body fluids such as ascites, pleural effusion, seroma, edema or wound drainage fluid are produced in large volumes, the concentration of tumor marker protein in such body fluids makes such fluids practical for antigens. There was concern whether it was high enough to allow it to be used as a source. Such body fluids are naturally expected to have a lower concentration of tumor marker protein compared to blood or serum. Surprisingly, the inventors have observed that the concentration of tumor marker protein in such body fluids is actually significantly higher than serum. Thus, considerable advantages are gained regarding the yield in recovering tumor marker proteins from such body fluids.

  In addition, it has also been observed by the inventors that pooling bodily fluid samples or excreta from two or more patients provides additional significant advantages. Besides increasing the protein yield, this product gives at least as good detection rate as the marker protein from the individual sample, and at the same time its features are more consistent between batches. It can therefore be relied on that the affinity of the antigen is sufficient each time.

  In certain embodiments, the methods of the invention may comprise an immunoassay that detects (simultaneously) two or more autoantibodies each having specificity for different tumor marker proteins or different epitopes on the same tumor marker protein. . These methods typically involve the use of a panel of two or more assay reagents, each reagent containing a different tumor marker protein. In these methods, hereinafter referred to as “panel assays”, two or more reagents are used to monitor an individual's overall immune response to tumors or other carcinogenic / neoplastic changes. Use the panel. Thus, these methods detect a “profile” of the immune response in a given individual that indicates which tumor marker elicits an immune response that results in the production of autoantibodies. The use of a panel of two or more reagents to monitor the production of autoantibodies against two or more different tumor markers is generally more sensitive than the detection of autoantibodies against a single marker and the frequency of false negative results Is much lower.

  The method of the invention is preferred for the detection of circulating free autoantibodies, but as will be appreciated by those skilled in the art, it is adapted for the detection of autoantibodies present in immune complexes, for example by competitive use of labeled tumor markers. obtain.

  In preferred applications, the methods of the invention are used to detect the presence of cancer-related anti-tumor autoantibodies in a human subject or patient, most preferably performed in vitro on a sample of body fluid taken from the subject / patient. Take the form of an immunoassay. Such in vitro immunoassays are non-invasive and can be considered as necessary to build a profile of autoantibody production in patients, such as in the screening of “at risk” individuals, before the onset of disease. Or can be repeated throughout the course of the disease (detailed below in connection with the preferred application of the method). The term “body fluid” as used herein with respect to substances to be tested by immunoassay for the presence of autoantibodies includes, among others, plasma, serum, whole blood, urine, sweat, lymph, feces, cerebrospinal fluid, ascites, pleural effusion , Semen, sputum or nipple aspirate. The type of bodily fluid used can vary depending on the type of cancer involved and the clinical situation in which the assay is used. In general, it is preferred to perform the assay on serum or plasma samples.

  As mentioned above, the “immunoassay” used to detect / quantify cancer-related autoantibodies can be performed according to standard techniques known in the art. In the most preferred embodiment, the immunoassay can be an ELISA. ELISA is generally well known in the art. In a typical “sandwich” ELISA, a reagent having specificity for the autoantibody under test is immobilized on a solid surface (eg, the well of a standard microtiter assay plate or the surface of a microbead) and A sample of body fluid to be tested for presence is contacted with the immobilized reagent. Any autoantibody of the desired specificity present in the sample will bind to the immobilized reagent. Any suitable method can then be used to detect the bound autoantibody / reagent complex. In one preferred embodiment, a labeled secondary anti-human immunoglobulin antibody that specifically recognizes an epitope common to one or more classes of human immunoglobulin is used to detect autoantibody / reagent complexes. . Typically, the secondary antibody is anti-IgG or anti-IgM. Secondary antibodies are usually labeled with a detectable marker, typically an enzyme marker such as peroxidase or alkaline phosphatase, thereby providing a substrate for the enzyme that produces a detectable product, such as a color, chemiluminescent or fluorescent product. Quantitative detection is possible by adding. Other types of detectable labels known in the art may be used and provide equivalent effects.

  The ELISA is performed in a qualitative format where the objective is simply to determine whether autoantibodies are present or absent in the sample, or in a quantitative format that provides a measure of the amount of autoantibodies present in the sample. obtain. Quantitative assays use the same detection reaction as used in the assay by measuring the signal obtained from a series of standard samples containing known concentrations of antibody with similar specificity as the autoantibody under test. Can) create a calibration curve. The amount of autoantibodies present in the sample under test can then be interpolated from the calibration curve.

  The panel assay is performed in a multiwell format in which each of two or more assay reagents are placed in separate wells of a multiwell assay plate, or a single pot format in which two or more assay reagents are placed in a single container. Can be done.

  The methods of the invention can be adapted for use to detect autoantibodies against essentially all tumor marker proteins. Appropriate “assay reagents” may be prepared from body fluids and / or excretions from body cavities of cancer patients. Specifically, the present method was performed using the epidermal growth factor receptor-related protein c-erbB2 (Dsouza, B. et al. (1993), Oncogene., 8: 1797-1806), glycoprotein MUC1 (Batra, S. K. et al. (1992), Int. J. Pancreatology., 12: 271-283) and the signal transduction / cell cycle regulatory protein Myc (Blackwood, EM et al., (1994), Molecular Biology of the Cell, 5: 597. -609), p53 (Matlashewski, G. et al. (1984), EMBO J., 3: 3257-3262; Wolf, D. et al. (1985), Mol. Cell. Biol., 5: 1887-1893) and ras (or Ras) (C apela, G. et al., (1991), Environ Health Perspectives., 93: 125-131), and BRCA1 (Scully, R. et al., (1997), PNAS, 94: 5605-10), BRCA2 (Sharan, S. et al.). K. et al., (1997), Nature., 386: 804-810), APC (Su, LK et al., (1993), Cancer Res., 53: 2728-2731; Munemitsu, S. et al., (1995). ), PNAS, 92: 3046-50), CA125 (Nouwen, EJ et al., (1990), Differentiation., 45: 192-8), PSA (Rosenberg, RS, et al., (1998), Biochem. Biophys Res C mun., 248: 935-939), carcinoembryonic antigen CEA (Duffy, MJ, (2001), Clin Chem, April, 47 (4): 624-30), and CA 19.9 (Haga, Y. et al., (1989), Clin Biochem, (1989) October, 22 (5): 363-8) may be applied to detect / measure autoantibodies. However, the present invention is not intended to be limited to the detection of autoantibodies against these specific tumor markers.

  The assay method of the present invention may be used in a variety of different clinical situations. Specifically, the method can be used to detect or diagnose cancer, monitor the progression of cancer or other neoplastic disease in a patient, change early neoplasia or change early carcinogenicity in an asymptomatic human subject. Detection, screening of a population of asymptomatic human subjects to identify subjects at high risk of developing cancer, monitoring the response of cancer patients to anti-cancer treatment, previously diagnosed with cancer It may be used in the detection of recurrent disease in patients who have received anti-cancer treatment to reduce the amount of cancer present, or in the selection of an anti-cancer vaccine for use in a particular patient.

  The present inventors generally observed that the level of cancer-related autoantibodies shows a positive correlation with the disease state (see International Publication No. WO99 / 58979, the contents of which are incorporated herein by reference). . Therefore, when the method of the present invention is used in clinical applications, an increase in the level of anti-tumor marker autoantibodies when compared to appropriate controls is taken as an indicator of cancer pathology.

  For example, when an immunoassay is used in the diagnosis of cancer, an increased level of autoantibodies compared to a “normal” control individual is taken as an indicator that the individual is afflicted with cancer. A “normal” control individual is a control that preferably has no diagnosis of cancer based on age-matched clinical, imaging and / or biochemical criteria.

  When an immunoassay is used to monitor the progression of cancer or other neoplastic disease in a patient, an increased level of autoantibodies compared to a “normal control” indicates that the patient has cancer. It is taken as an indicator. A “normal control” may be the level of autoantibodies present in a control individual that has no diagnosis of cancer, preferably based on age-matched clinical, imaging and / or biochemical criteria. Alternatively, the “normal control” can be the “reference” level established for the particular patient under study. The “reference” level can be, for example, the level of autoantibodies that are present when an initial cancer diagnosis or a recurrent cancer diagnosis is made. Any increase above the baseline level is taken as an indicator that the amount of cancer present in the patient has increased, while any reduction below the baseline is taken as an indicator that the amount of cancer present in the patient has been reduced. . Also, the “reference” value can be, for example, a level before starting a new treatment. Changes in the level of autoantibodies are taken as an indicator of treatment effectiveness. The direction of “change” (ie, increase or decrease) that indicates a positive response to treatment depends on the detailed nature of the treatment. For any treatment, the direction of the “change” in autoantibody levels that gives a positive result can be readily determined. This is done, for example, by monitoring autoantibody levels relative to other clinical or biochemical indicators of response to treatment.

  When immunoassays are used to screen a population of asymptomatic human subjects to identify subjects at high risk of developing cancer, levels of autoantibodies are increased compared to “normal” control individuals The individual is identified as being “at risk” to develop cancer. A “normal” control individual is preferably an age-matched control that has been identified as having no predisposition to developing cancer or having no significant increase in risk of developing cancer. An exception to this may be when age itself is a major risk factor.

  When immunoassays are used to monitor a cancer patient's response to an anti-cancer treatment, a decrease in autoantibody levels after treatment is taken as an indication that the patient has responded positively to the treatment. The baseline level of autoantibodies obtained before initiating treatment can be used for comparative purposes to determine whether the treatment results in a “reduction” in autoantibody levels.

  When immunoassays are used to detect recurrent disease, an increased level of patient autoantibodies compared to a “normal control” is taken as an indication that the disease has recurred. A “normal control” can be the level of autoantibodies present in a control individual that has no diagnosis of cancer, preferably based on age-matched clinical, imaging and / or biochemical criteria. Alternatively, the “normal control” can be the “reference” level established for the particular patient under study. The “reference” level can be, for example, the level of autoantibodies present during the remission phase of the disease based on clinical, imaging and / or biochemical criteria.

  The assay methods of the present invention can be applied to the detection of many different types of cancer, such as breast cancer, bladder cancer, colon cancer, prostate cancer and ovarian cancer. The assay may supplement existing screening and research methods. For example, in the case of primary breast cancer, autoantibody immunoassays can be used to perform biopsies on small mammogram lesions that do not appear suspicious on radiography, or to perform breast imaging or imaging earlier than scheduled. It can alert the clinician to repeat. In the clinic, the assay method of the present invention is expected to be more objective and reproducible than current imaging techniques (ie, mammography and ultrasound) whose success may depend on the operator.

A “panel assay” can be tailored for a particular clinical application. A panel of reagents for detecting autoantibodies against at least p53 and c-erbB2 is particularly useful for many types of cancer and is known to be associated with the particular cancer to be detected or with a particular period of cancer to be detected. Other markers can be optionally added. For example, for breast cancer, the panel may include at least one of MUC1, c-myc, BRCA1, BRCA2, and PSA, and for bladder cancer, the panel optionally includes at least one of MUC1 and c-myc. And at least one of ras and APC for colon cancer, at least one of PSA, BRCA1 and BRCA2 for prostate cancer, or at least one of BRCA1, BRCA2 and CA125 for ovarian cancer . There are other preferred embodiments in which p53 or c-erbB2 is not necessarily essential. For example, for breast cancer, an appropriate panel can be selected from:
p53 and MUC1, and optionally at least one of c-erbB2, c-myc, BRCA1, BRCA2 and PSA;
p53 and c-myc and optionally at least one of c-erbB2, MUC1, BRCA1, BRCA2 and PSA;
p53 and BRCA1, and optionally at least one of c-erB2, MUC1, c-myc, BRCA2 and PSA;
p53 and BRCA2, and optionally at least one of c-erbB2, MUC1, c-myc, BRCA1 and PSA;
c-erbB2 and MUC1, and optionally at least one of p53, c-myc, BRCA1, BRCA2 and PSA;
c-erbB2 and c-myc and optionally at least one of p53, MUC1, BRCA1, BRCA2 and PSA;
c-erbB2 and BRCA1, and optionally at least one of p53, MUC1, c-myc, BRCA2 and PSA;
c-erbB2 and BRCA2, and optionally at least one of p53, MUC1, c-myc, BRCA1 and PSA.

For colorectal cancer, an appropriate panel can be selected from:
p53 and ras and optionally at least one of c-erbB2 and APC;
p53 and APC and optionally at least one of c-erbB2 and Ras;
Ras and APC and optionally at least one of p53 and c-erbB2.
Such panels may also include CEA or CA19-9.

For prostate cancer, an appropriate panel can be selected, for example, from:
p53 and PSA and optionally at least one of BRCA1, BRCA2 and c-erbB2;
c-erbB2 and PSA and optionally at least one of p53, BRCA1 and BRCA2.

In the case of ovarian cancer, an appropriate panel can be selected, for example, from:
p53 and CA125 and optionally at least one of c-erbB2, BRCA1 and BRCA2;
c-erbB2 and CA125, and optionally at least one of p53, BRCA1 and BRCA2.

  In further embodiments, the immunoassay methods of the present invention may be used in the selection of anti-cancer vaccines for use in specific patients. In this embodiment, in order to determine the relative strength of a patient's immune response to each of the different tumor marker proteins, a sample of bodily fluid collected from the patient is taken with two or more immunoassay reagents each corresponding to a different tumor marker protein. Test using the panel. The “intensity of immune response” for a given tumor marker protein or proteins is indicated by the presence and / or amount of cancer-related autoantibodies specific for that tumor marker protein detected using an immunoassay. . When quantifying autoantibodies, the higher the level of cancer-related autoantibodies, the stronger the immune response. Subsequently, one or more tumor marker proteins identified to elicit the most potent immune response or responses (ie, highest autoantibody levels) in the patient are selected for use in the patient Form the basis for vaccines.

  In a further embodiment, the present invention monitors whether vaccination of a subject with an anti-cancer vaccine based on a particular tumor marker protein has succeeded in eliciting a humoral immune response (ie, an antibody against said tumor marker protein) Provide a method. This method consists of an immunoassay method used to measure cancer-related anti-tumor autoantibodies (ie, use of an immunoassay reagent based on a tumor marker protein purified from body cavity fluid or excreta taken from a cancer patient) and Based on the same method, the only difference is that what is measured in the assay is the antibody response, not the autoantibody response.

In this embodiment, previously treated with an anti-cancer vaccine (eg, an immunogenic preparation comprising a relevant tumor marker protein or antigenic fragment thereof, or a vaccine comprising a nucleic acid encoding said relevant tumor marker protein) A sample of body fluid taken from a patient is contacted with an immunoassay reagent to detect complexes formed by specific binding between the immunoassay reagent and a cancer-related antibody present in the sample. Again, immunoassay reagents, one or more samples of tumor marker protein prepared from the body fluid of the body 腔由 come as defined herein for cancer patients, and / or one or a plurality of cancer patients Contains a sample of tumor marker protein prepared from feces.

  In addition to clinical use in detecting cancer and the like, the methods of the invention may be used in any application where it is desired to test for the presence of cancer-related anti-tumor autoantibodies. For example, the method of the present invention may have application as a research tool in a laboratory.

  The tumor marker protein preparation provided by the present invention is advantageously used as (component) of an immunoassay reagent used in the assay method of the present invention. However, the usefulness of tumor marker protein preparations is not limited to such use. For example, these can also be used as research tools in the laboratory. Furthermore, tumor marker proteins can have utility as therapeutic agents. The availability of large amounts of protein provided by body fluids as defined herein allows in vitro or in vitro in human or non-human animals to determine the effectiveness of a particular tumor marker protein as a therapeutic agent Vivo preclinical and clinical trials will be possible. Such test methods apply to each and all of the various tumor marker proteins described herein.

  Another usefulness of the tumor marker preparations of the present invention is as a calibrator for use in conjunction with the development of a diagnostic test for the presence or risk of cancer, which test can be performed in any patient clinical sample. Based on determining the presence and / or level of certain tumor marker proteins. The tumor marker protein preparation of the present invention can be used to construct a calibration curve for such a test. Specifically, this aspect of the invention includes:

A method of calibrating an assay for measuring or detecting a predetermined tumor marker protein in a clinical sample comprising:
a) preparing at least two samples of tumor marker protein prepared according to the method of the present invention, each comprising said predetermined tumor marker protein, each having a different concentration of tumor marker protein than each of said other said samples Steps,
b) i) a spectrophotometric or spectrophotometric method, and / or ii) performing a quantitative measurement of the concentration of the tumor marker protein in each of the samples using an antibody reagent against the tumor marker protein;
c) constructing a standard curve of the concentration of the tumor marker protein based on the measured value obtained in step (b).

  Such a calibration curve may be constructed for any or all of the specific tumor marker proteins described herein.

  The invention will be further understood with reference to the following experimental examples in conjunction with the accompanying figures.

Example 1 General Protocol for Purifying MUC1 Antigen Monoclonal anti-MUC1 antibody B55 (NCRC11, also known as Xoma Corporation) is conjugated to CNBr-Sepharose beads. Instead of B55, another anti-MUC1 monoclonal antibody may be used.

  Tumor-induced fluid (eg pleural effusion, ascites, seroma or wound drainage fluid) is diluted 1/10 with phosphate buffered saline (PBS) and filtered at 0.45 μm.

  Incubate diluted body fluid with anti-MUC1 Sepharose beads (1 ml filled volume of beads for 25 ml of diluted body fluid) overnight at 4 ° C. with rotation (“batch” method) or anti-MUC1 Sepharose beads Recirculate in a packed column containing (“column” method).

“Batch” method beads are filled by centrifugation and the supernatant is removed.
The beads are resuspended in 5-10 ml PBS and spun for 10 minutes, after which they are filled by centrifugation and the supernatant removed. Repeat 5 times (or until A _280nm ˜0).
Resuspend the beads in 5 ml 100 mM DEA, pH 11, and spin at room temperature for 10 minutes.
Fill the beads by centrifugation to remove the supernatant, adjust the pH to 7 by adding pH 7 Tris buffer and dialyze against PBS for a minimum of 24 hours (100 DEA fraction).
The beads are resuspended in 5 ml PBS and spun for 10 minutes, after which the beads are filled by centrifugation to remove the supernatant, the pH is adjusted to 7 by adding pH 7 Tris buffer, and a minimum of 24 Dialyze against PBS for time (post-DEA fraction).
Prior to pooling these two fractions and storing at -20 ° C., the MUC1 content of each fraction was measured using, for example, the monoclonal anti-MUC1 antibody C595 (available from Cancer Research Campaign Laboratories, UK) 5)) or confirmed by ELISA using B55.

The “column” method column is washed with 5 column volumes of PBS or until the eluate reading is about 0 at A _{280 nm} .
Run 1 column volume of 100 mM DEA, pH 11, then 5 column volumes of PBS.
The eluate fraction is collected from the time of flowing DEA to the time of flowing PBS (2 ml).
The fraction is dialyzed overnight against PBS.
Fractions are assayed for MUC1 content by ELISA using, for example, monoclonal anti-MUC1 antibody C595 or B55, and positive fractions of MUC1 are pooled and stored at -20 ° C.
To remove contaminating immunoglobulin, the pooled fraction of MUC1 was incubated with dithiothreitol (DTT) for up to 50 mM for 30 minutes, followed by incubation with iodoacetamide (up to 75 mM) followed by gel filtration on an S300 column. Do.
The resulting fraction (5 ml) is assayed by ELISA for MUC1 and human immunoglobulin (Ig) content.
MUC1-containing fractions (not contaminated with human Ig) are pooled and stored at -20 ° C.

Example 2a General protocol for purifying MUC16 antigen (formerly known as CA125) 1 volume (eg 50 ml) of saturated ammonium sulphate 1 volume (eg 50 ml) of tumor-induced body fluid (eg pleural effusion, ascites) , Seroma or wound drainage fluid) and incubated overnight at 4 ° C.
The resulting precipitate is collected by centrifugation (standard bench top centrifuge for 30 minutes at 3500 rpm) and resuspended in 1/2 volume of PBS.
This resuspension is subjected to gel filtration chromatography on an S300 column (2.5 × 100 cm) using PBS as the elution buffer.
Fractions (5 or 10 ml) were collected and assayed for MUC16 by ELISA using, for example, ICN anti-CA125 or anti-MUC16 antibody VK8 (Memorial Slane Kettering, NY) and the positive fraction of MUC16 was pooled to −20 Store at ℃.
To remove contaminating immunoglobulin, the pool of MUC16 is incubated with NaSCN (up to 1.5M) for 10 minutes, with DTT (up to 50 mM) for 30 minutes, then with iodoacetamide (up to 75 mM), then for example S300 Alternatively, gel filtration is performed on a Superdex ™ 75 column.
The resulting fraction (5 ml) is assayed by ELISA for MUC16 and human immunoglobulin (Ig) content.
Fractions containing MUC16 (not contaminated with human Ig) are pooled and stored at -20 ° C.

Example 2b Post- Ig Breaking Gel Filtration Chromatography Samples prepared in the manner described above, fractions from Ig post-breaking gel filtration were collected for MUC16 using anti-MUC16 antibody VK8 and human using anti-human Ig. Assayed for Ig. The results are shown in FIG. As clearly shown, two MUC16 peaks that are substantially free of immunoglobulin are eluted.

(Example 3) Purification of c-myc antigen The MUC1 purification method (Example 1) is followed except for the following.
Monoclonal anti-c-myc antibody 9E10 (ATCC) is used (or equivalent anti-c-myc antibody).
Gel filtration is performed on a Superdex ™ 75 column.

Electrophoresis and Western Blot Purity of MUC1, MUC16 and c-myc fractions was determined using a BioRad ™ Mini Protean III ™ system and a DryBlot ™ system according to standard protocols according to denaturing polyacrylamide gel electrophoresis. Assess by electrophoresis and Western blot.
Silver staining revealed a protein pattern of c-myc on the gel (FIG. 2). The Western blot of c-myc was immunoprobed using monoclonal antibody 9E10 (FIG. 3). In each case, c-myc and immunoglobulin heavy and light chains are identified.

Example 4 Standard Autoantibody Assay Tumor antigen appropriately diluted in PBS (eg, MUC1, MUC16 or c-myc prepared according to Examples 1-3) in a standard 96 well microtiter plate 50 μl per well And air-dried overnight.
The plate is washed once with PBS / Tween ™ to remove residual salt crystals.
Plates are blocked for 60 minutes with 0.1% casein or 1% BSA in PBS.
Wash plate 3 times with PBS / Tween ™.
Serum (diluted 1/100 with PBS / 0.1% casein) in triplicate (50 μl per well) and monoclonal antibody control.
Incubate for 60 minutes at room temperature with shaking.
Wash plate 4 times with PBS / Tween ™.
Horseradish peroxidase (HRP) -conjugated anti-Ig antibody (Dako) is added to each well (50 μl per well) at a dilution of 1/8000 for anti-human and 1/1000 for anti-mouse. Add.
Incubate for 60 minutes at room temperature with shaking.
Wash plate 4 times with PBS / Tween ™.
50 μl TMB (tetramethylbenzazine) was added per well and read kinetically at A _{650 nm} for 10 minutes.

Experimental Data Using the method described in Example 4, cancer-related autoantibodies against MUC1 and MUC16 were isolated in various sera using MUC1 and MUC16 isolated from the various sources described herein. Measured with The obtained results are shown in FIGS.

  FIG. 4 compares the reactivity of a patient's serum autoantibodies to MUC1 isolated from various body fluids: urine (from “normal” individuals), pleural effusion from cancer patients and serum from advanced breast cancer patients (ABC serum). FIG. The patient sera tested are from individuals with primary breast cancer who do not show any signs of breast cancer but have a family history of breast cancer (ie, one or more relatives suffered from breast cancer at a young age) It was derived from an individual.

  A standard autoantibody ELISA was performed as described above, using MUC1 isolated from urine (normal), pleural effusion or ABC serum as an antigen. Data was normalized with an internal control reaction using DF3 anti-MUC1 monoclonal antibody (compared to serum sample) for each of the MUC1 antigens.

  As can be seen from the figure, normal urine (nMUCl) derived MUC1 was consistently less reactive than pleural effusion (PE) or ABC serum derived MUC1. Furthermore, PE-derived MUC1 was similar to MUC1 isolated from the sera of patients suffering from ABC for reactivity to cancer-related MUC1 autoantibodies, and thus had similar diagnostic values.

  FIG. 5 shows the results of the same exercise as FIG. 4 except that all tested serum samples were from normal individuals (no breast cancer or family history of breast cancer). As can be seen from the figure, there is no significant difference in the reactivity of sera to three different antigens.

  FIG. 6 shows the reactivity of MUC16 cancer-related autoantibodies from the sera of patients suffering from ovarian masses (before surgery) to MUC16 (CA125) isolated from normal individual sera and ascites from breast cancer patients. FIG. Antigen was prepared as in Example 2, and autoantibodies were detected using the ELISA assay described in Example 4.

  As can be seen from the figure, it can be seen that the reactivity of cancer-related MUC16 autoantibodies is greatly enhanced by the MUC16 antigen derived from ascites as compared to “normal” MUC16. Therefore, the results of this experiment confirmed the usefulness of ascites as an antigen source for detecting cancer-related autoantibodies.

Example 5 Measurement of cancer-related MUC1 levels in ascites, pleural effusions, seroma and wound drainage fluid Patients with serum samples collected in each case were MUC1 levels found in the sera of patients suffering from cancer Compared to levels found in ascites, pleural effusion, wound drainage or seroma in the same patient. MUC1 in the sample was quantified according to the following protocol.

Capture MUC1 ELISA protocol 50 μl of antibody solution per well is distributed into triplicate wells of microtiter plates (usually 1 μg ml ⁻¹ C595 (IgG) and appropriate negative control) and incubated for 1 hour with shaking at room temperature And adsorb the protein to the plate.
Wash the plate 4 times with PBS / Tween using 250 μl per well.
Block the plate with 100 μl per well of 1% BSA and incubate for 1 hour with shaking at room temperature.
Wash the plate 4 times with PBS / Tween using 250 μl per well.
Add 50 μl of body fluid to be tested per well (diluted 1/10 with PBS) and incubate for 1 hour with shaking at room temperature.
Wash the plate 4 times with PBS / Tween using 250 μl per well.
Add 50 μl biotinylated C595 (1 μg / ml) per well and incubate for 1 hour with shaking at room temperature.
Wash the plate 4 times with PBS / Tween using 250 μl per well.
Add 50 μl extra avidin peroxydice at a dilution of 1/1000 per well and incubate for 1 hour with shaking at room temperature.
Wash the plate 4 times with PBS / Tween using 250 μl per well.
50 μl TMB substrate was added per well and read dynamically at 650 _nm for 10 minutes.

  The results are shown in FIGS.

  As can be readily seen from the data, serum levels of cancer-associated MUC1 antigen were significantly lower than those found in any of ascites, pleural effusion, seroma or wound drainage fluid. Therefore, recovering tumor marker antigens from these body cavity fluids provides significant benefits in terms of yield.

Example 6 Reactivity of Human Anti-MUC1 Antibody Purified against Serumoma-Derived Cancer-Related MUC1 Serumoma-derived human antibody of patient M was subjected to immunoaffinity chromatography to seroma of the same cancer patient M. Purified against the derived MUC1. The purified antibodies were then tested against MUC1 protein core peptide conjugated to BSA and MUC1 from patient M seromas collected after diagnosis of patient M urine collected after 2 years of cancer diagnosis. Purification of the antibody from seroma was performed according to the following protocol.

Purification of human anti -MUC1 antibody Purification of human anti-MUC1 autoantibody was performed by affinity chromatography.
Seroma fluid diluted 10-fold with PBS, pH 7.6 was loaded onto the affinity matrix in the form of a column consisting of CNBr Sepharose (Pharmacia) conjugated to Pt-MUCl (according to the manufacturer's instructions) and 0.5 ml / Recirculated at 4 ° C. overnight in minutes.
After the seroma fluid is applied, the column is washed with 15 ml of PBS (to ensure that the A ₂₈₀ nm reading returns to 0) and the antibody is eluted with 10 ml of 3M NaSCN at 1 m / min. It was.
A 1 ml fraction was collected throughout, desalted by dialysis against PBS, and tested for the presence of antibody by ELISA.
Positive fractions were pooled, antibody purity was confirmed (by PAGE), and antibody concentration was determined.
Purified antibody assays against the three MUC1 antigens identified above were performed according to the following protocol.

MUC1 ELISA protocol 50 μl per well of MUC1 antigen solution is dispensed into triplicate wells of a microtiter plate and dried overnight at room temperature.
The plate is washed twice with PBS / Tween using 250 μl per well.
Block the plate with 100 μl per well of 1% BSA and incubate for 1 hour with shaking at room temperature.
The plate is washed twice with PBS / Tween using 250 μl per well.
Add 50 μl of purified antibody solution at 1 μg / ml per well and incubate for 1 hour with shaking at room temperature.
Wash the plate 4 times with PBS / Tween using 250 μl per well.
Add 50 μl of α-human Ig HRP (DAKO) per well diluted freshly according to the manufacturer's instructions and incubate for 1 hour with shaking at room temperature.
Wash the plate 4 times with PBS / Tween using 250 μl per well.
Add 50 μl TMB substrate per well and read dynamically at 650 _nm for 10 minutes.

  The results are shown in FIG.

  The reactivity of the antibody against the MUC1 peptide was negligible. The reactivity of the antibody to normal MUC1 was significantly lower than that seen against MUC1 from patient M seroma. From this result, it can be deduced that normal MUC1 molecules differ substantially from those found in seroma fluids from individuals suffering from cancer with regard to their immune recognition.

Example 7 Reactivity of Serum to MUC1 Purified from Pooled Ascites and Pleural Fluid Purified from pooled ascites and pooled pleural effusions of patients suffering from advanced breast cancer using the protocol described in Example 1 The reactivity to sera from patients suffering from primary breast cancer was measured as described in Example 4. Antigens from pooled bodily fluids were compared with antigens isolated from three separate samples of ascites or pleural effusions in patients each suffering from ABC in each case. The results are shown in FIGS.

  In both cases of ascites and pleural effusion, the reactivity of MUC1 from pooled bodily fluids was as good as that isolated from individual samples. In addition, there is a wide range of variability in reactivity for the use of samples from individuals, but pooled samples provide greater consistency of products, so that reactivity varies significantly between batches of pooled samples. Is unthinkable.

Example 8 Calibration curve using MUC1 A serial dilution of MUC1 isolated from pleural effusion was prepared. These MUC1 concentrations were measured by the method shown in Example 4 except that human serum was not used. Detection was performed with mouse B55 antibody followed by Dako anti-mouse HRP, reading the end point rather than a dynamic reading.

  The results are shown in FIG. 12, which confirms the usefulness of the tumor marker protein prepared according to the present invention as a calibration substance.

Sources of antibodies against tumor marker proteins The sources of antibodies that can be used for purification of tumor marker proteins by affinity chromatography are shown below. Affinity chromatography can be performed according to the general method described in Example 1 (for MUC1) with appropriate modifications. It will be appreciated that other antibodies specific for the relevant marker protein may also be used.

Carcinoembryonic antigen (CEA):
1116NS-3d, ATCC No. CRL-8019, a monoclonal antibody producing B lymphocyte hybridoma against CEA; T84.66A3.1A. Monoclonal antibody-producing B lymphocyte hybridoma against 1F2, ATCC No. HB-8747, CEA.

P53:
Rabbit anti-human p53 polyclonal, commercially available from Serotec Ltd, Kidlington, Oxford OX5 1JE, UK.
Monoclonal anti-p53, clone BP53-12, commercially available from Sigma.

CA19-9:
Mouse anti-human CA19-9 monoclonal, clone 1116-NS-19-9 type, IgG1, commercially available from Serotec Ltd, Kidlington, Oxford OX5 1JE, UK.

H-ras p21:
Santa Cruz Biotechnology, Inc. Rabbit polyclonal IgG commercially available from Santa Cruz, California, USA.

BRCA1:
Santa Cruz Biotechnology, Inc. Rabbit polyclonal IgG commercially available from Santa Cruz, California, USA.

BRCA2:
Santa Cruz Biotechnology, Inc. Goat polyclonal IgG, commercially available from Santa Cruz, California, USA.

APC:
Santa Cruz Biotechnology, Inc. Rabbit polyclonal IgG commercially available from Santa Cruz, California, USA.

PSA:
Santa Cruz Biotechnology, Inc. Mouse monoclonal IgG commercially available from Santa Cruz, California, USA.

FIG. 7 shows a post-Ig disruption gel filtration chromatogram of an ascites-derived MUC16 (CA125) preparation. It is a figure which shows the silver-stained gel of the purified c-myc derived from ascites after immunoaffinity chromatography. It is a figure which shows the immunoprobing blot of purified c-myc derived from ascites after immunoaffinity chromatography. Patient's serum (the person does not show any signs of breast cancer) against various body fluids, ie urine (from "normal" individuals), pleural effusion from cancer patients and serum from advanced breast cancer patients (ABC serum) FIG. 7 shows a comparison of autoantibody reactivity in patients with a family history of breast cancer and patients suffering from primary breast cancer. FIG. 5 shows the reactivity of autoantibodies in serum from normal individuals against various body fluids, urine MUC1 (normal), pleural effusion-derived MUC1 from cancer patients and advanced breast cancer patients (ABC serum). FIG. 5 shows the reactivity of autoantibodies in serum samples from preoperative patients suffering from ovarian masses against normal MUC16 (CA125) from ascites and tumor-associated MUC16. FIG. 5 shows cancer-related MUC1 concentrations in serum, pleural effusion and ascites. FIG. 7 shows the concentration of cancer-related MUC1 in serum, wound drainage fluid and seroma. MUC1 of patient M's purified urine, MUC1 derived from seroma of patient M, collected after 2 years of diagnosis of cancer, post-diagnostic and against bovine serum albumin conjugated with MUC1 protein nucleopeptide FIG. 3 shows the reactivity of purified autoantibodies derived from seroma of patient M. FIG. 2 shows the reactivity of serum autoantibodies to MUC1 purified from pooled ascites and to MUC1 purified from individual ascites samples of cancer patients. FIG. 2 shows the reactivity of serum autoantibody reactivity to MUC1 purified from pooled pleural effusion and to MUC1 purified from individual pleural effusion samples of cancer patients. It is a figure which shows the calibration curve created from MUC1 derived from pleural effusion.

Claims (14)

A method for detecting cancer-related antitumor autoantibodies, comprising:
Contacting a sample to be tested for the presence of such autoantibodies with an immunoassay reagent, and specific binding of said immunoassay reagent and any cancer-related anti-tumor autoantibodies present in said sample. An immunoassay comprising detecting the presence of a complex
The immunoassay reagent, the one or more cancer patients, the tumor had to or present there, or tumor related to that or related to have body 腔由 come tumor marker protein prepared from bodily fluid The tumor marker protein exhibits selective reactivity with a cancer-related antitumor autoantibody,
The method wherein the autoantibodies are present at elevated levels in cancer patients compared to normal control individuals. An immunoassay to detect and / or quantitatively determine the presence of two or more autoantibodies, each immunologically specific for two or more epitopes of a different tumor marker protein or the same tumor marker protein Including doing,
Wherein the immunoassay, at least one reagent comprises one or more cancer patient's body 腔由 come tumor marker protein prepared from bodily fluid is carried out using a panel of two or more immunoassay reagents, wherein Item 2. The method according to Item 1. The sample to be tested using the immunoassay is a sample of body fluid collected from the patient;
An increased level of autoantibodies compared to a normal control individual is taken as an indicator that the individual is suffering from or is developing cancer,
3. A method according to claim 1 or claim 2 for detecting cancer in a patient. The sample to be tested using the immunoassay is a sample of body fluid collected from the patient;
An increase in the level of autoantibodies compared to normal controls is taken as an indicator of the presence of cancer in the patient,
3. A method according to claim 1 or claim 2 for monitoring the progression of cancer or other neoplastic disease in a patient. The sample to be tested using the immunoassay is a sample of body fluid collected from the subject;
The increased level of autoantibodies compared to normal control individuals is taken as an indicator of early neoplastic or early carcinogenic changes in the subject,
3. A method according to claim 1 or claim 2 for detecting early neoplastic or early carcinogenic changes in an asymptomatic subject. The sample to be tested using the immunoassay is a sample of body fluid collected from the subject;
An increased level of the subject's autoantibodies compared to normal control individuals is identified as being at risk of developing cancer,
3. A method according to claim 1 or claim 2 for screening a population of asymptomatic human subjects to identify subjects at high risk for developing cancer. The sample to be tested using the immunoassay is a sample of body fluid collected from the patient;
A decrease in autoantibody levels after treatment is taken as an indicator that the patient responded positively to the treatment,
3. A method according to claim 1 or claim 2 for monitoring the response of cancer patients to anti-cancer therapy. The patient is receiving anti-cancer treatment to reduce the amount of cancer present,
The sample to be tested using the immunoassay is a sample of body fluid collected from the patient;
An increase in the level of autoantibody in the patient compared to a normal control is taken as an indicator that the disease has recurred,
3. A method according to claim 1 or claim 2 for detecting a recurrent disease in a patient who has already been diagnosed as having cancer. The immunoassay is performed using a panel of two or more immunoassay reagents, each corresponding to the different tumor marker protein to determine the relative strength of the patient's immune response to each of the different tumor marker proteins;
Selecting one or more tumor marker proteins identified to elicit the most potent immune response or responses in the patient to form the basis of an anti-cancer vaccine for use in the patient;
The method of claim 2 for the selection of an anti-cancer vaccine for use in a particular patient. A vaccination procedure comprising administering to a patient an immunogenic preparation comprising a tumor marker protein or antigenic fragment thereof or a nucleic acid sequence expressing said tumor marker protein, wherein said patient is a cancer-related antibody against said tumor marker protein A method to determine whether or not
Contacting a sample of the patient's bodily fluid with an immunoassay reagent, and detecting the presence of a complex formed by the specific binding of the immunoassay reagent to any cancer-related antibody present in the sample. An immunoassay comprising:
Wherein the immunoassay reagent is one or more cancer patients, the tumor was to or present there, or tumor related to that or tumor marker protein prepared associated have body lumen or al specimen ,
The tumor marker protein shows selective reactivity with anti-tumor antibodies of related cancer,
A method wherein the presence of the antibody at an elevated level in the patient compared to a normal control individual indicates the success of the vaccination procedure . The body 腔由 come fluid is ascites, pleural effusion, seroma, a Mizukobu or wound drainage fluid, A method according to any one of claims 1 to 10.   12. The method of claim 11, wherein the tumor marker protein is selected from MUC1, MUC16 or c-myc.   The method according to any one of claims 1 to 11, wherein the tumor marker protein is selected from c-erbB2, p53, ras, BRCA1, BRCA2, APC, PSA, CEA, and CA 19.9. Shows selective reactivity with anti-tumor autoantibodies related cancer, wherein the autoantibodies compared to normal control individuals present at elevated levels in cancer patients in the manufacture of an immunoassay reagent, one or more of cancer patients, the tumor had to or be present there, or tumor is related to that or related to have use of the tumor marker protein prepared from the body 腔由 come of body fluids.

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